In recent years, studies of culturing animal or plant cells under various conditions and studies of products of cultured cells have been actively carried out. Particularly, studies on production of substances, of which artificial synthesis is impossible or very difficult, utilizing certain cell activities are carried out in various fields. Also studies are carried out to identify substances that affect cellular growth and differentiation so as to obtain proliferation or differentiation of certain cells with them according to the purpose. Also with the rapid progress in cell engineering and medical engineering, minute biosensors, artificial organs, neurocomputers and the like are attracting attentions and actively studied.
In order to utilize cells in vitro as explained above, it is essential to dispose cells to control their proliferation, differentiation and substance production in a desired manner. However, the mechanisms of cell disposition, cell proliferation and differentiation, and substance production have not been sufficiently clarified, so that the cell culture under controlled conditions is extremely difficult impeding researches utilizing cells.
Also tailor-made therapy considering individual difference in the sensitivity to drugs, of which concept has recently been widely recognized, is strongly desired, but the influence of biologically active substances has been investigated on the function of respective substances, particularly because of technical reasons, and there has not been established an effective method of easily investigating presence/absence of effects of plural drugs, or a required dose thereof or a combined effect thereof.
As an attempt to control cell disposition, U.S. Pat. No. 6,368,838 discloses a method for culturing cells by forming a pattern of cell adhesive portions on a substrate to control arrangement of cells of plural species. U.S. Pat. No. 5,108,926 also describes formation of a pattern on a substrate by applying a cell-adhering protein using an ink jet printer and cell culture thereon. These methods allow to culture cells on the formed pattern of the cell-adhering protein, but not to control the proliferation, differentiation and substance production of the cells or to achieve screening of cell culture conditions. Also “Protein, Nucleic acid and Enzyme”, vol. 45, 727-734 (2000) describes immobilization of a cell growth factor, which affects cell proliferation or differentiation, on a substrate using photolithography, thereby investigating its influence on the cell proliferation and differentiation. However, the substrate on which the cell growth factor is immobilized is not used for screening, and the photolithography has problems that a rare biological substance is wasted and the production process is complicated requiring repetition of exposure and development steps.
Japanese translation of PCT international application No. 2000-512009 proposes a cell screening method by immobilizing onto a substrate a substance that affects cell adhesion. In this method, reactive functional groups provided on the substrate and the cell-adhering substance are bonded through a divalent crosslinking reagent, and photolithographic technology is used to bond the reactive functional group and the cell-adhering substance. However it has a problem, in addition to the aforementioned problems, that when plural cell-adhering substances are immobilized, it is extremely difficult to avoid a situation that an already immobilized substance and a substance to be newly immobilized are bonded by the divalent crosslinking reagent in undesired positions, that is, it is extremely difficult to arrange cell-adhering substances in desired positions. Also the proposed method is not to immobilize a substance influencing the cell proliferation, differentiation and substance production. That method is to screen cells by immobilizing cells in individual wells through the immobilized adhering substance, culturing the cells in a culture medium and detecting a certain substance produced by the cells. Thus it is not intended for screening a substance which influences at least one of adhesive property, proliferation, differentiation, survival, maintenance of an undifferentiated state, death and material production of cells, as intended in the present invention.
U.S. Pat. No. 6,103,479 discloses a method that allows effective confirmation of effects of a physiologically active substance using cells, where different cells are immobilized on a substrate to form an array to which an physiologically active substance is applied. This method, however, does not consider immobilization of physiologically active substances onto a substrate. This method requires a microfluidic delivery system to delivery the physiologically active substance to the substrate, and when plural physiologically active substances are used, complicated operations are required to load them onto the apparatus.
Also Japanese Patent Application Laid-open No. 2002-355025 discloses a method of forming a screening substrate characterized in immobilizing plural screening substances by using liquid discharge means in desired areas of a base, thereby providing different screening functions. In this invention, since the substances for screening are immobilized to the screening substrate, cells often cannot intake the screening substance into the cells. Therefore, this invention is not effective in the case where only the screening substance taken into the cells can affect at least one of the cellular functions including proliferation, differentiation, survival, maintenance of an undifferentiated state, death and substance production.
Also Japanese Patent Application Laid-open No. 2002-328124 discloses a screening method with a higher order combination of biological active substances, but this is to evaluate an effect of a function of biological active substances provided to a substrate, so it cannot be used for screening effects of biological active substances that function in vivo in a state immobilized on the extracellular matrix, or an effect induced by successive addition of biological active substances.
Also Japanese Patent Application Laid-open No. 2003-33177 proposes a simple assay of chemical substances such as drugs or toxic substances, preparing a cell array divided into plural areas and providing a biologically active substance to each area to carry out simultaneous screening of plural samples. In such a method, however, each biologically active substance is provided to the cultured cells by using a dispensing means. Thus there is a risk of contamination in the dispensing step and it requires a specific apparatus for dispensing the biologically active substances, far from convenient use.